11/14/2006 – HCV Therapy – Pre-Clinical and Early Clinical Development

 

 

Abstract ID: 67405

Category: JO6: HCV Therapy: Preclinical and Early Clinical Development

Therapeutic vaccination for chronic hepatitis C virus infection in HCV trimera mice.

F. Rahman, I. Department of Internal Medicine, University Mainz, Germany, Mainz,

Germany, N. Blust, I. Department of Internal Medicine, University Mainz, Germany,

Mainz, Germany, T. Baumert, Internal Medicine II,, Freiburg, Germany, M. Geissler,

Internal Medicine II,, Freiburg, Germany, P. R. Galle, I. Department of Internal

Medicine, University Mainz, Germany, Mainz, Germany, W. O. Böcher, I. Department

of Internal Medicine, University Mainz, Germany, Mainz, Germany

 

Discussion

Whereas spontaneous resolution from hepatitis C is associated with strong antiviral T cell responses in the acute phase of the disease, only weak immune responses are found in patients developing chronic infection. Thus, induction of strong HCV-specific T cell responses could represent a new therapeutic strategy. Virus like particles (VLP) expressing the HCV structural proteins as well as synthetic peptides representing well characterized CTL epitopes might serve as an effective therapeutic vaccine. The humanized trimera mouse model was used to study the immunogenicity of these vaccine candidates in a preclinical animal model. Irradiated Balb/c mice were transplanted with nod.scid mouse bone marrow and reconstituted with human blood lymphocytes (PBMC) from three HLA A2.1 positive chronic HCV patients. These so-called trimera mice were vaccinated intraperitoneally with VLP expressing the structural proteins core, E1 and E2 or with a mixture of HLA A2 restricted synthetic peptide CTL epitopes from the HCV core, E1, E2 or NS3 region. PBMC were recovered 10 days after vaccination and T cell analysis was performed with tetramer staining or gIFN Elispot assay with recombinant HCV antigens, overlapping HCV peptides or the respective CTL epitopes. HCV specific T cell responses could be detected in trimera mice reconstituted with PBMC from two out

of three chronic HCV patients, although such responses were undetectable in donor PBMC. Alternatively, instead of PBMC reconstitution, HCV-infected human liver tissue was implanted under the kidney capsule of the trimera mice. HCV quantification in the trimera serum and HE-staining of implanted liver grafts was performed at different time points. Viremia was detectable in several mice for more than three weeks after implantation and histological staining showed viable liver tissue up to this point. Since the trimera mouse model supports both, induction of viremia and immune responses, it should allow preclinical testing of new vaccine candidates, such as lipopeptides,

exosomes or dendritic cells, making a therapeutic vaccination a potentially successful approach for the future.


 

Abstract ID: 64638

Category: JO6: HCV Therapy: Preclinical and Early Clinical Development

 

Demonstration of the Efficacy of Small Molecule HCV Inhibitors in the KMT MouseModel of Hepatitis C Virus (HCV) Infection.

N. Kneteman, University of Alberta Hospital, Edmonton, Canada

 

 

Aim

We report efficacy of clinically proven inhibitors of HCV infection (Interferon alpha 2b, BILN 2061) in our mouse model, thus demonstrating the utility of the model for HCV antiviral drug discovery.

 

MATERIALS AND METHODS

SCID carrying the urokinase type plasminogen activator gene (KMT mice) were transplanted with human hepatocytes to yield mice with chimeric human livers (Nature Medicine 7:927, 2001). Mice with HCV titres104-107 copies/ml received 1350 IU/gram body INFalpha,(n=18 X 2 wk; n=4 X 4 wk) or saline (n=16 X 2 wk; n=4 X 4 wk) by IM daily. Nine mice received 10mg/kg bid orally of the protease inhibitor BILN2061for 4 (n=3) or 7(n=6) days. Mice with increasing titre (after initial drop) despite ongoing treatment with BILN2061 had RT-PCR products from plasma/liver sequenced to evaluate development of viral escape mutations as previously reported in the replicon system during BILN 2061 administration.

 

RESULTS

INF resulted in a consistent decrease in viral titre of 0.7, 1.1, and 2.0 log after 1, 2 and 4 wk (P<0.01 at all time points vs control). Therapy with BILN2061 resulted in viral titres dropping mean 2 log after 4 and 1.2 log after 7 days (P<0.0001 BILN2061 vs. vehicle, P<0.03 /BILN vs. INF). The D168V mutation previously reported to be associated with resistance to BILN2061 in replicon cultures (J. Virol. (2003) 77:3669) was not found in the sera or liver. Pre-existing mutants (Q80K) with partial resistance to

BILN2061 were identified.

 

CONCLUSIONS

Results with INF and BILN2061 therapy of HCV infection in the KMT mouse model of HCV infection parallel reported outcomes in clinical application (Nature 426:186, 2003), validating the KMT model for evaluation of potential anti-HCV therapeutics. HCV escape mutations previously reported during BILN2061 treatment of HCV replicons in vitro were not seen in this in vivo system. This system should help advance into human trials, lead compounds with an increased likelihood of clinical success while broadening the tools available for study of the biology of HCV infection.


 

Abstract ID: 65035

Category: JO6: HCV Therapy: Preclinical and Early Clinical Development

 

Pharmacokinetics, Safety, and Tolerability of the Isatoribine Oral Prodrug ANA975 in aPhase 1 Healthy Volunteer Study.

 

B. Kerr, Anadys Pharmaceuticals Inc, San Diego, CA, L. Bauman, Anadys

Pharmaceuticals, Inc., San Diego, CA, S. Webber, Anadys Pharmaceuticals, Inc., San

Diego, CA, A. Xiang, Anadys Pharmaceuticals, Inc., San Diego, CA, J. Ng, Anadys

Pharmaceuticals, Inc., San Diego, CA, L. Kirkovsky, Anadys Pharmaceuticals, Inc., San

Diego, CA, D. Bartkowski, Anadys Pharmaceuticals, Inc., San Diego, CA, K. Steffy,

Anadys Pharmaceuticals, Inc., San Diego, CA, S. Fletcher, Anadys Pharmaceuticals,

Inc., San Diego, CA, R. Aust, Anadys Pharmaceuticals, Inc., San Diego, CA, J. Theiss,

SunCoast Tox, San Diego, CA, D. Averett, Anadys Pharmaceuticals Inc, San Diego, CA

 

PURPOSE

In a proof-of-concept (POC) clinical study, intravenous (IV) infusion of the

TLR7 agonist isatoribine 800mg once daily x 7 days to patients chronically infected with hepatitis C virus (HCV) yielded a significant reduction of plasma HCV RNA that correlated with induction of 2’, 5’-oligoadenylate synthetase (OAS). Oral doses of isatoribine are poorly absorbed in multiple species. The prodrug ANA975 was developed to improve oral delivery of isatoribine to systemic circulation.

 

METHODS

Toxicology studies, including GLP oral multiple-dose studies in cynomolgus monkeys, were performed to enable clinical studies. An open label, rising

dose level, single-dose study in healthy volunteers was performed in the United Kingdom according to institutional and local regulatory requirements. Thirty-six subjects each received a single oral dose of ANA975 administered as a 5mg/mL solution in the fasted state at doses of 400mg (n=6), 800mg (n=18), or 1200mg (n=12).

 

RESULTS

Oral administration of ANA975 to monkeys resulted in efficient systemic

delivery of isatoribine; the highest tolerated dose in monkey achieved plasma area-underthe-curve (AUC) values 6-13x higher than achieved with the 800mg IV dose in the clinical POC study. Monkeys showed a dose-related OAS induction in blood that persisted with once daily dosing for treatment periods up to at least 28 days. Single oral doses of ANA975 were well tolerated by healthy volunteers. There were no serious adverse events (AEs) and no withdrawals from the study. A total of 15 AEs were reported by 13 of 36 subjects. All reported AEs were mild (13 events) or moderate (2

events) in severity. The Investigator considered 1 AE (mild transient increase in CPK at 800mg) to be possibly related to ANA975 and all other AEs to be unlikely related or not related to ANA975. There were no reports of gastrointestinal AEs or flu-like syndrome. ANA975 was rapidly and extensively converted to isatoribine, with isatoribine Cmax typically occurring within 1 hour post-dose. At the highest tested dose, which has an

isatoribine dose content of 988mg, plasma isatoribine AUC values (range 23-40, median 27mg*h/L) were similar to those observed in the clinical POC study with daily 1-hour IV infusions of isatoribine 800mg (first dose AUC range 19-38, median 24mg*h/L). The similarity of AUC values at comparable molar doses of oral ANA975 and IV isatoribine indicates that fractional oral absorption and conversion of ANA975 to isatoribine was very high.

 

CONCLUSION

ANA975 is an efficient oral prodrug that achieves plasma isatoribine exposures comparable to those previously shown to reduce plasma HCV RNA in chronically infected patients.  The data is encouraging and support further studies of ANA975.


Abstract ID: 60761

Category: JO6: HCV Therapy: Preclinical and Early Clinical Development

 

Decline in serum neopterin concentration correlates with HCV RNA decline during administration of VX-950, a Hepatitis C Virus Protease Inhibitor.

H. C. Gelderblom, Academic Medical Center, Department of Gastroenterology and Hepatology, Amsterdam, Netherlands, S. Zeuzem, Saarland University Hospital, Homburg/Saar, Germany, C. J. Weegink, Academic Medical Center, Department of Gastroenterology and Hepatology, Amsterdam, Netherlands, N. Forestier, Saarland University Hospital, Homburg/Saar, Germany, L. McNair, Vertex Pharmaceuticals, Cambridge, MA, S. Purdy, Vertex Pharmaceuticals, Cambridge, MA, P. L. Jansen, Academic Medical Center, Department of Gastroenterology and Hepatology, amsterdam, Netherlands, H. W. Reesink, Academic Medical Center, Department of Gastroenterology and Hepatology, Amsterdam, Netherlands

 

Background

Neopterin is a guanosine triphosphate (GTP) derived compound that is produced by activated monocytes/macrophages. We followed neopterin levels during administration of VX-950, an orally administered inhibitor of the Hepatitis C virus (HCV) NS3·4A protease, in a multiple-dose study in 34 patients chronically infected with HCV genotype 1.

 

Methods

VX-950 was administered for 14 days at doses of 450 mg or 750 mg every 8

hours, or 1250 mg every 12 hours, or placebo. Serum neopterin levels were measured by immunoassay at days -1, 7 and 14 of dosing, and day 10 of follow-up (day 24). HCV RNA was assessed by real-time PCR.

Results

In patients with chronic HCV infection, VX-950 had substantial antiviral

effects, with every patient demonstrating at least a 2-log drop in viral load in all dosing groups. In the 750 mg q8h dose group, there was a reduction in median HCV RNA of more than 3 log10 after 3 days, and of 4.4 log10 at the end of 14 days of dosing. In the 450 mg q8h and 1250 mg q12h dose groups, maximal effect was seen between 3 and 7 days of dosing followed by an increase in median viral load between days 7 and 14. Median viral loads increased in all groups between days 14-24 (post-dosing). Baseline neopterin

levels were elevated in 23/34 patients (median 9.45 nmol/l; ULN 7.7 nmol/l). In the 750 mg q8h dose group, the changes from baseline in median neopterin level were -3.6, -3.8 and -1.3 nmol/l at days 7, 14 and 24 (figure). In the 450 q8h dose group, the changes were -1.8, -1.4 and +0.4 nmol/l at days 7, 14 and 24 (figure). In the 1250 q12h dose group, the changes were, +0.3, -0.2 and + 1.0 nmol/l at days 7, 14 and 24 (figure). In the placebo group, the changes from baseline in median neopterin level were -0.2, +0.5, and +0.4 nmol/l at days 7, 14 and 24. Median ALT levels, which were elevated at baseline, normalized during 14 days of dosing in all groups.

 

Discussion

Changes in median neopterin levels correlated with the decrease in HCV

RNA and ALT during administration of VX-950. The maximal decrease in median neopterin level was in the 750 mg q8h dose group, which was also the dose group with maximal reductions in HCV RNA. These data suggest that inhibition of HCV replication by VX-950 abrogates inflammation.

 


 

Abstract ID: 66459

Category: JO6: HCV Therapy: Preclinical and Early Clinical Development

Characterization of NM283 (Valopicitabine) Resistance Profile Using Bovine Viral Diarrhea Virus.

V. Bichko, Idenix Pharmaceuticals, Inc., Cambridge, MA, L. Qu, Idenix

Pharmaceuticals, Inc., Cambridge, MA, M. La Colla, Idenix Pharmaceuticals, Inc.,

Cambridge, MA, M. Tausek, Idenix Pharmaceuticals, Inc., Cambridge, MA, S.

Bergelson, Idenix Pharmaceuticals, Inc., Cambridge, MA, C. Pierra, Laboratoire

Coopératif Idenix-CNRS-Université Montpellier II, Montpellier, R. Storer, Idenix

Pharmaceuticals, Inc., Cambridge, MA, G. Gosselin, Laboratoire Coopératif Idenix-

CNRS-Université Montpellier II, Montpellier, J. Sommadossi, Idenix Pharmaceuticals,

Inc., Cambridge, MA, D. Standring, Idenix Pharmaceuticals, Inc., Cambridge, MA

 

 

Background

Valopicitabine (NM283) reduces serum hepatitis C virus (HCV) RNA

levels in patients and in chronically infected chimpanzees, and is currently in phase II clinical development for the treatment of chronic hepatitis C. NM283 is an orally bioavailable prodrug of NM107 (2’-C-methylcytosine), which inhibits flavi- and pestivirus replication in vitro and shows enhanced antiviral activity in combination with interferon alfa. The present study evaluated the potential for the development of resistance to NM283 using NM107 and a bovine viral diarrhea virus (BVDV) in vitro infection model. BVDV is a pestivirus related to HCV.

 

Methods

NM107-resistant BVDV variants were isolated by passaging infected DBK

cells with varying concentrations of NM107. Both cytopathic (cp) and noncytopathic (ncp) BVDV biotypes were evaluated. Resistant mutants were characterized in terms of replication fitness (see below) and sensitivity to NM107 and Intron A. A mutation within the NS5B region was confirmed by sequencing.

 

Results

Repeated passaging of a cp strain of BVDV (NADL) with NM107 failed to

produce resistant mutants. However, although prolonged NM107 treatment can eradicate BVDV in a cell line persistently infected with ncp BVDV, lower concentrations of NM107 led to selection of resistant virus after 3 to 5 cell passages. Sequencing revealed resistance was due to a S405T amino acid substitution near the start of the B domain motif of the BVDV NS5B polymerase, confirming that the polymerase is the target for NM107. This motif is highly conserved among positive-strand RNA viruses, including

HCV, predicting that the equivalent mutation in HCV polymerase is S282T. The NM107- resistant BVDV strains show at least 50-fold reduced susceptibility to NM107, accompanied by decreased replication fitness in vitro: “small plaque” phenotype, slow growth kinetics and lower virus titers. Importantly, the S405T mutant virus showed ~38 fold increased susceptibility to IFN a-2b compared to wild type BVDV. Consistent with

this finding, no viral resistance developed in vitro when NM107 was used in combination with Intron A. To date, we have been unable to find additional compensatory mutations that restore the phenotype of the S405T mutant BVDV to that of wild type.

 

Conclusions

Resistance of BVDV to NM107 (due to the S405T mutation in NS5B) was associated with reduced replication fitness, a lack of compensatory  mutations, and increased sensitivity to Intron A. The homologous mutation in HCV NS5B is S282T. However, this mutant (or NM283 resistance) has yet to be observed in the clinic after up to 6 months therapy with interferon alfa 2b and NM283 in the ongoing phase II clinical trial.


Abstract ID: 66647

Category: JO6: HCV Therapy: Preclinical and Early Clinical Development

REDIRECTING T LYMPHOCYTE SPECIFICITY USING HCV-SPECIFIC T CELL RECEPTOR GENES CONFERS RECOGNITION OF NATURALLY OCCURRINGVIRAL MUTANTS.

 

H. R. Rosen, PVAMC, Portland, OR, G. Callender, U. Chicago, Chicago, IL, G. E.

Lyons, University of Chicago, Chicago, IL, M. Nishimura, U. Chicago, Chicago, IL

 

Background: HCV infection is manifested by a high rate of viral persistence related to high viral mutability as well as diminished CTL effector function. We have previously identified a T cell receptor (TCR) that has relatively high affinity for HLA A2-restricted HCV NS3 1406-1415 peptide. The purpose of this study was to determine whether gene transfer of this TCR into peripheral blood lymphocytes (PBL) from normal healthy controls could induce HCV-specific reactivity that would respond to wild-type and

mutant virus.

 

Methods

TCR alphaƒnand beta chain genes from the HCV-specific TCR were cloned

and inserted into a retroviral vector capable of delivering the genes into human T cells. The ability of transferred TCR genes to confer HCV reactivity to PBL from 3 healthy donors was evaluated using IFN-gamma production following stimulation with wild type or mutant peptide.

 

Results

TAP-deficient T2 cells were loaded with wild type NS3 1406-1415 peptide

(KLVALGINAV), eight different naturally occurring viral mutants (V1408L, A1409T, I1412V, I1412L, I1412N, V1408T and the multiple substituted peptides A1409G/I1412L/V1408T and  1408S/A1409G/I1412L), or a melanoma control peptide. TCR-transduced PBL (both CD4+ and CD8+ T cells) produced statistically significant amounts of IFN-gamma following stimulation with wild type HCV peptide and 7 of the 8 HCV mutant peptides but not with melanoma peptide. Moreover, TCR-transduced PBL recognized endogenously processed peptide.

 

Conclusions

We demonstrate the ability to engineer normal PBL-derived T cells to

specifically recognize HCV independent of immune status. By providing T cell help as well as effector function, this approach represents a promising immunotherapy. Moreover, the ability of TCR-transduced PBL to recognize mutants suggests this approach might prevent antigenic escape variants.


 

Abstract ID: 66787

Category: JO6: HCV Therapy: Preclinical and Early Clinical Development

Single Dose Pharmacokinetics of a Novel Hepatitis C Protease Inhibitor, SCH 503034, in an Oral Capsule Formulation.

J. Zhang, Schering-Plough Research Instutute, Kenilworth, NJ, S. Gupta, Schering-

Plough reserach Institute, kenilworth, NJ, R. Rouzier, Centre Cap, Montpelleir, France,

A. Calzetta, Schering-Plough Research Institute, Kenilworth, NJ, D. L. Cutler,

Schering-Plough Research Institute, Kenilworth, NJ

 

Background

SCH 503034, is an orally active Hepatitis C virus NS3 serine protease inhibitor that exhibits potent and specific in vitro antiviral activity. We report results from our Phase I clinical development program evaluating the pharmacokinetics (PK) and safety of SCH 503034 oral capsules in healthy subjects.

 

Methods

In this randomized, single rising dose, double-blind study, healthy subjects were randomized 2:1 to receive, SCH 503034 oral capsules, (n=6/group ) 50 mg, 100 mg, 200 mg, 400 mg, 600 mg, 800 mg or placebo (n=3/group). Plasma samples were collected at multiple time points to 120h post-dose. Samples were analyzed by validated LC-MS/MS with a LOQ of 0.5 ng/ml. Pharmacokinetics were analyzed using model independent methods. Safety was assessed by clinical laboratory tests, vital signs, ECGs, physical exams and occurrence of adverse events.

 

Results

Fifty-four subjects completed the study, 36 received SCH 503034, 18 received placebo. SCH 503034 was rapidly absorbed following oral administration of capsules (mean Tmax :1-2.25 h across the 6

dosing levels). After attaining Tmax, plasma SCH503034 concentrations declined in a bi-phasic manner, with a mean terminal phase half-life (T1/2 ) of 7.0 to 15 h. Cmax and AUC increased in a dose-related manner. The safety profiles were similar in subjects receiving SCH 503034 and placebo.

 

Conclusions

SCH 503034 was readily bioavailable when administered as a single dose in an oral capsule formulation and was well tolerated in subjects receiving up to 800 mg orally.

 


 

Abstract ID: 59834

Category: JO6: HCV Therapy: Preclinical and Early Clinical

Development Identification of a Novel Small Molecule Hepatitis C Virus Replication Inhibitor that Targets Host Sphingolipid Biosynthesis.

H. Sakamoto, Kamakura Research Laboratories, Chugai Pharmaceutical Co. Ltd.,

Kamakura, Japan, K. Okamoto, Kamakura Research Laboratories, Chugai

Pharmaceutical Co. Ltd., kamakura, Japan, M. Aoki, Kamakura Research Laboratories,

Chugai Pharmaceutical Co. Ltd., Kamkura, Japan, H. Kato, Kamakura Research

Laboratories, Chugai Pharmaceutical Co. Ltd., Kamakura, Japan, A. Katsume,

Kamakura Research Laboratories, Chugai Pharmaceutical Co. Ltd., Kamakura, A. Ohta,

Kamakura Research Laboratories, Chugai Pharmaceutical Co. Ltd., Kamakura, Japan, T.Tsukuda, Kamakura Research Laboratories, Chugai Pharmaceutical Co. Ltd., Kamakura, Japan, N. Shimma, Kamakura Research Laboratories, Chugai Pharmaceutical Co. Ltd.,Kamakura, Japan, Y. Aoki, Kamakura Research Laboratories, Chugai Pharmaceutical Co. Ltd., Kamakura, Japan, M. Arisawa, Kamakura Research Laboratories, Chugai Pharmaceutical Co. Ltd., Kamakura, Japan, M. Kohara, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan, M. Sudoh, Kamakura Research Laboratories, Chugai Pharmaceutical Co. Ltd., Kamakura, Japan

 

Introduction

An estimated 170 million individuals worldwide are infected with hepatitis C virus (HCV), a serious cause of chronic liver disease. Current interferon-based therapy for treating HCV infection has an unsatisfactory cure rate, and the development of more efficient drugs is needed. Here, we have identified a lipophilic long-chain base compound, NA255, from a secondary fungal metabolite, as a novel small molecule HCV replication inhibitor using an HCV subgenomic replicon cell culture system. HCV replicon 1b cell-based assay showed that the replication was suppressed by NA255 in a dose-dependent manner with a mean of 50% inhibitory concentration (IC50) of 2 nM. NA255 had no effect on host-cell viability (IC50>50,000 nM) and cell cycle progression. We found that NA255 prevents the de novo synthesis of sphingolipids, major lipid raft components, thereby inhibiting serine palmitoyltransferase and disrupting the association among HCV nonstructural (NS) viral proteins on the lipid rafts. To determine whether

HCV protein could interact directly with sphingolipids, we searched for the sphingolipidbinding domain (SBD) in HCV NS protein. We found that NS5B protein has a SBD in the molecular structure and that the interaction of NS5B with sphingolipids could be involved in HCV replicon replication.

 

Conculsion

Thus, NA255 is a potent anti-HCV replication inhibitor that targets host lipid raft, suggesting that inhibition of sphingolipid metabolism may provide a novel therapeutic strategy for treatment of HCV infection.


 

Abstract ID: 60227

Category: JO6: HCV Therapy: Preclinical and Early Clinical

Development SCH 503034, a Mechanism-based Inhibitor of Hepatitis C Virus (HCV) NS3 Protease Suppresses Polyprotein Maturation and Enhances the Antiviral Activity of Interferon-á-2b (INF)

B. A. Malcolm, Schering-Plough Research Instutute, Kenilworth, NJ, A. Arassappan,

Schering Plough Research Institute, Kenilworth, NJ, F. Bennett, Schering-Plough

research Institute, Kenilworth, NJ, S. Bogen, Schering-Plough research Institute,

Kenilworth, NJ, R. Chase, Schering-Plough research Institute, Kenilworth, NJ, K. Chen,

Schering-Plough research Institute, Kenilworth, NJ, T. Chen, Schering-Plough research

Institute, Kenilworth, NJ, P. Ingravallo, Schering-Plough research Institute, Kenilworth,

NJ, E. Jao, Schering-Plough research Institute, Kenilworth, NJ, S. Kong, Schering-

Plough research Institute, Kenilworth, NJ, F. Lahser, Schering-Plough research Institute,

Kenilworth, NJ, R. Liu, Schering-Plough research Institute, Kenilworth, NJ, Y. Liu,

Schering-Plough research Institute, Kenilworth, NJ, R. Lovey, Schering-Plough research

Institute, Kenilworth, NJ, J. McCormick, Schering-Plough research Institute,

Kenilworth, NJ, G. F. Njoroge, Schering-Plough research Institute, Kenilworth, NJ, A.

Saksana, Schering-Plough research Institute, Kenilworth, NJ, A. Skelton, Schering-

Plough research Institute, Kenilworth, NJ, X. Tong, Schering-Plough research Institute,

Kenilworth, NJ, S. Venkatraman, Schering-Plough research Institute, Kenilworth, NJ, J.

Wright-Minogue, Schering-Plough research Institute, Kenilworth, NJ, E. Xia, Schering-

Plough research Institute, Kenilworth, NJ, V. Girijavallabhan, Schering-Plough research

Institute, Kenilworth, NJ

 

Background

Cleavage of the HCV polyprotein by the NS3 protease releases proteins essential for viral propagation. Additionally, NS3-mediated cleavage of host factors integral to Interferon Response Factor-3 signaling may result in abrogated cellular responses to IFN. Thus, blockage of NS3 activity is expected to inhibit HCV replication by direct suppression of viral production and restoration of host IFN responsiveness. SCH 503034, reversibly binds the HCV NS3 protease and specifically inhibits its activity.

 

Methods

SCH 503034 NS3 binding activity was determined spectrophotometrically. Anti-viral potency was assayed in HCV replicon cells exposed to increasing amounts of SCH 503034 for various times up to 15d. HCV replicon RNA and protein expression were monitored by Northern and Western analysis, respectively and replicon copy number was determined by qRT-PCR

multiplex reaction. The effect of SCH 503034 on response to IFN was evaluated in replicon cells incubated with increasing amounts of SCH 503034 and IFN for 72h. Toxicity, was assessed by cell viability and doubling time.

 

Results

SCH 503034 exhibited time-dependent inhibition of single chain NS3 with a binding constant (Ki*) equal to ~14 nM. SCH 503034 inhibition of replicon synthesis was dose-related. The IC50 and IC90 for suppression of replicon synthesis following 72-h exposure were ~200 nM and 400 nM, respectively. Exposure of replicon cells to SCH 503034 over 15d resulted in log

reductions in replicon RNA of ~ 2.0, 3.5, and 4.0 at 1.2, 6 and 12 x IC90, respectively. Isobologram analysis revealed that SCH 503034 + IFN-

α was more effective than the line of additivity, though the difference was not significant (p>0.1). No toxicity was seen after exposure of HuH-7 cells to 50μM SCH 503034 for 96 h, or primary hepatocytes to 10 x IC90 for 1 wk. No changes in doubling times or growth rates of HuH-7 cells were seen after a 3-wk at 10 x IC90.

Conclusions

SCH 503034 exhibited potent anti-viral activity both alone and in combination with IFN-α in the HCV replicon assay. The combination of SCH 503034 with IFN may lead to enhanced clinical efficacy.

 


 

Abstract ID: 67060

Category: JO6: HCV Therapy: Preclinical and Early Clinical Development

HCV replication is inhibited by cyclosporin A, mycophenolic acid and IFN-alpha in a synergistic fashion and with distinct anti-viral kinetics.

S. Henry, Erasmus MC, Rotterdam, Netherlands, H. Metselaar, Erasmus MC,

Rotterdam, Netherlands, R. Bartenschlager, Otto-Meyerhof Center, Heidelberg,

Germany, H. Tilanus, Erasmus MC, Rotterdam, Netherlands, L. van der Laan, Erasmus

MC, Rotterdam, Netherlands

 

Background

Chronic Hepatitis C Virus (HCV) infection is the leading cause of liver transplantation worldwide. The success of transplantation, however, is compromised by the re-infection of the graft and this problem has begun to worsen in the last decade. Changes in immunosuppressive therapy may have

contributed to this deterioration. The aim of this study is to determine the effect of different immunosuppressive compounds on HCV replication and determine the synergy and kinetics of inhibition.

 

Methods

The anti-viral properties of pegylated IFN-alpha 2b (IFN) and immunosuppressive drugs were tested in vitro using an HCV-replication model. Huh-7 hepatoma cells, containing the HCV replicon with a

luciferase reporter gene, were cultured for 18 hour with different doses or combinations of drugs. HCV replication was quantified based on luciferase activity, RT-PCR for viral RNA and immunocytochemistry for helicase (NS3) protein. Kinetics of inhibition was determined with the Xenogen IVIS imaging system.

 

Results

Tacrolimus, rapamycin, dexamethason or prednisolon did not inhibit HCV replication, whereas almost complete inhibition (98%) was observed with IFN (10IU/ml). Both cyclosporin A (CsA) (0.5-5.0 ug/ml) and mycophenolic acid (MPA) (2.0-6.0 ug/ml) blocked HCV replication up to 70%. No cell death was observed with any tested compounds. Proliferation of Huh-7 cells was reduced with MPA, but did not account for the observed inhibition of HCV replication. When CsA and MPA were combined, synergistic inhibition was observed up to 90% at higher doses. Synergistic inhibition was also observed with a combination of CsA and suboptimal doses of IFN. The anti-viral kinetics of CsA, MPA and IFN were shown to be different (See Fig).

 

Conclusion

The immunosuppressive drugs CsA and MPA both are potent and specific inhibitors of HCV replication. The synergy and different anti-viral kinetics suggest that antiviral activity of both drugs act via independent pathways and can operate independent of each other. These findings suggest that

immunosuppressive therapy based on a combination of CsA and MPA (low or free of steroids) is preferable for reducing HCV recurrence after transplantation.

 


 

Abstract ID: 61330

Category: JO6: HCV Therapy: Preclinical and Early Clinical Development

NIM811 exhibits potent anti-HCV activity in vitro and represents a novel approach for viral hepatitis C therapy.

K. Lin, Novartis Institutes of Biomedical Research, Cambridge , MA, S. Ma, Novartis

Institutes of Biomedical Research, Cambridge, MA, J. Boerner, Novartis Institutes of

Biomedical Research, Cambridge, MA, M. Huang, Novartis Institutes of Biomedical

Research, Cambridge, MA, N. Ryder, Novartis Institutes of Biomedical Research,

Cambridge, MA, B. Weidmann, Novartis Institutes of Biomedical Research, Cambridge,

MA, M. P. Cooreman, Novartis, East Hanover, NJ

 

Introduction/ Discussion

Host factors required in the lifecycle of hepatitis C virus (HCV) are potential targets of

antiviral therapy, and are complementary to inhibitors of viral enzymes such as NS3-4A

protease and NS5B polymerase. The anti-HCV effect of CsA has been demonstrated both

in vitro and in a controlled clinical trial. Various CsA analogs were evaluated for their

inhibitory effect on viral RNA replication in HCV replicon cells. There was a good

correlation between antiviral activity and cyclophilin-binding activity of these

compounds. NIM811 was selected for further research because it binds to cyclophilins

with similar affinity as CsA but is devoid of significant immunosuppressive activity, as

the NIM811-cyclophilin complex does not bind to calcineurin. NIM811 induced a

concentration-dependent reduction of HCV RNA in replicon cells with an IC50 of 0.66

ìM at 48 hours, compared to 0.80 ìM for CsA. After a nine-day treatment of replicon

cells, a three-log10 reduction of HCV RNA levels was achieved with as low as 1 ìM

NIM811. Moreover, NIM811 showed equally potent activity in HCV replicons that were

highly resistant to viral protease or polymerase inhibitors. Resistance against this

compound appears to be difficult to generate in vitro. In vitro, combinations of NIM811

with other HCV inhibitors produced at least additive antiviral effects. Pharmacokinetic

studies in rats showed a t1/2 of circa 14 hours, with hepatic concentrations 10-fold over

serum levels. Toxicology studies showed no safety issue at the dose range predictive of

therapeutic efficacy. In conclusion, NIM811 has potent anti-HCV activity and is several

orders of magnitude less immunosuppressive than CsA in vitro. The fact that it targets a

host protein and the demonstration of its uncompromised efficacy in HCV resistant

against protease or polymerase inhibitors provide the rationale for combining of NIM811

with other specific antivirals to improve therapeutic efficacy and to suppress emergence

of drug-escape mutants.


 

Abstract ID: 62612

Category: JO6: HCV Therapy: Preclinical and Early Clinical Development Genetic heterogeneity in the HCV NS3 protease of untreated genotype 1 patients has little effect on the sensitivity to VX-950.

T. Kieffer, Vertex Pharmaceuticals Inc., Cambridge, MA, C. Sarrazin,

Universitätsklinikum des Saarlandes, Homburg, Germany, D. Bartels, Vertex

Pharmaceuticals Inc., Coralville, IA, B. Hanzelka, Vertex Pharmaceuticals Inc.,

Coralville, IA, U. Muh, Vertex Pharmaceuticals Inc, Coralville, IA, M. Welker,

Universitätsklinikum des Saarlandes, Homburg, Germany, D. Wincheringer,

Universitätsklinikum des Saarlandes, Homburg, Germany, C. Lin, Vertex

Pharmaceuticals Inc., Cambridge, MA, T. Grossman, Vertex Pharmaceuticals Inc.,

Cambridge, MA, C. Weegink, Academic Medical Center (AMC), Department of

Gasteroenterology and Hep, Amsterdam, Netherlands, S. Purdy, Vertex Pharmaceuticals

Inc, Cambridge, MA, H. Reesink, Academic Medical Center (AMC), Department of

Gasteroenterology and Hep, Amsterdam, Netherlands, A. Kwong, Vertex

Pharmaceuticals Inc, Cambridge, MA, S. Zeuzem, Universitätsklinikum des Saarlandes,

Homburg, Germany

 

Introduction/Discussion

The non-structural (NS)3-4A protease is essential for hepatitis C virus (HCV) replication and a promising target for new anti-HCV therapy. VX-950, a potent and specific NS3-4A protease inhibitor, has recently demonstrated significant antiviral activity in a phase 1b trial of patients infected with HCV genotype 1 . Median reductions of 2 to 4 log10 IU

per mL in serum HCV RNA concentrations were achieved after 14 days of dosing with VX-950. The degree to which a patient responds to treatment and the rate at which viral rebound is observed could in part be due to genotypic differences in sensitivity to the protease inhibitor. The rapid replication rate of hepatitis C virus, along with the poor fidelity of its polymerase, gives rise to an accumulation of mutations throughout its

genome. The degree to which sequence variability in the protease region affects the catalytic efficiency of the enzyme or the binding of an inhibitor is not known. Here, we characterize the extent of sequence diversity within the NS3 protease domain of HCV isolated from 34 patients enrolled in the phase 1b trial, before dosing with VX-950. The NS3 protease domain was amplified by nested RT-PCR, cloned, and sequenced. The consensus sequence for each patient’s HCV population was derived from an average of

90 independent cDNA clones. The average intra-patient amino acid quasispecies complexity (Shannon entropy) and diversity (Hamming distance) was low (0.332 ± 0.109 and 0.421 ± 0.195, respectively) and no correlation of quasispecies heterogeneity with HCV RNA serum concentration at baseline was observed. The inter-patient (individual

consensus compared to genotype 1a or 1b consensus sequence) amino acid diversity was 1% for genotype 1a and 2% for genotype 1b. Modeling analysis predicted that amino acid differences observed between consensus sequences of all these patients were expected to have little or no impact on VX-950 binding. Patient specific protease clones were then expressed and tested for inhibition by the protease inhibitor VX-950. In agreement with the modeling observation, there were no significant differences in the

enzymatic IC50 of these proteases derived from different patient isolates.

 

Conclusion

It appears that despite the observed sequence diversity in the HCV NS3 serine protease, patients may be uniformly responsive to treatment with the protease inhibitor VX-950.


 

Abstract ID: 67450

Category: JO6: HCV Therapy: Preclinical and Early Clinical Development

CANNABINOIDS BLOCK INTERFERON-MEDIATED SUPPRESSION OF HEPATITIS C VIRUS (HCV) REPLICATION.

S. Ramesh, Virginia Commonwealth University, Richmond, VA, F. Mirshahi, Virginia

Commonwealth University, Richmond, VA, J. Choudhury, Virginia Commonwealth

University, Richmond, VA, A. Sanyal, Virginia Commonwealth University, Richmond,

VA

 

BACKGROUND

Many patients with HCV infection use cannabinoids. Cannabinoid

receptor activation has both direct effects on hepatocytes and T-cell function. These could affect the virologic response to HCV treatment. The potential effects of cannabinoids on interferon-mediated suppression of viral replication were not known.

 

SPECIFIC AIMS

To define the effects of tetrahydrocannabinol (THC) and methanandamide (MA) a CB receptor agonist on interferon-mediated suppression of

HCV replication in hepatocytes.

 

METHODS

HCV virus replication was measured by negative strand quantitative PCR in

a commercially available HCV replicon system (APATH). The effects of increasing doses of MA (0-3 &#956;M) and THC (0-1.5 &#956;M) on HCV replication was first measured. Next, the effect of MA and THC on &#945;-interferon mediated suppression of HCV replication was measured. The specificity of these effects were tested by pharmacologic blockade of CB1 and CB2 receptors. Next, the effects of MA on interferon-mediated increased on protein kinase R (PKR) and interferon response factor

(IRF) mRNA (measured by qPCR), and protein (measured by Western blot) were measured.

 

RESULTS

Compared to controls, MA and THC produced a 40-62% decrease in HCV

replication. These effects were most marked after 12 hours of exposure. As expected, IFN (5000 IU/ml) produced a 90% inhibition of HCV replication. MA as well as THC blocked the IFN-mediated inhibition of HCV replication. These effects could be blocked by the CB1 receptor antagonist SR 141716 but not by a CB2 antagonist SR144528. MA and THC also blocked the interferon-mediated increase in PKR and IRF mRNA and

protein.

 

CONCLUSIONS

THC and other CB1 receptor agonists can block the IFN-mediated

suppression of HCV replication in a replicon system by inhibiting IFN-induction of IRF and PKR. The clinical relevance of these data should be assessed by correlation of cannabinoid use with virologic response to IFN therapy.


 

Abstract ID: 67430

Category: JO6: HCV Therapy: Preclinical and Early Clinical Development

Itmn A and B, novel inhibitors of the HCV NS3/4 protease retain their potency against VX-950 and BILN-2016 resistant NS3/4 protease mutants and an IFN-a-2a resistant HCV replicon.

S. Seiwert, InterMune, Brisbane, CA, S. W. Andrews, Array Biopharma, Boulder, CO,

H. Yang, Gilead, Foster City, CA, H. Tan, InterMune, Brisbane, CA, B. Marifino,

InterMune, Brisbane, CA, R. Radhakrishnan, InterMune, Brisbane, CA, E. Cheung,

InterMune, Brisbane, CA, R. Rieger, Array Biopharma, Boulder, CO, Y. Jiang, Array

BioPharma, Boulder, CO, A. Kennedy, Array BioPharma, Boulder, CO, S.

Wenglowsky, Array BioPharma, Boulder, CO, M. Madduru, Array BioPharma, Boulder,

CO, J. Bencsik, Array BioPharma, Boulder, CO, M. Liang, Array BioPharma, Boulder,

CA, B. Woodward, Array BioPharma, Boulder, CO, K. Condroski, Array BioPharma,

Boulder, CO, C. Lemieu, Array BioPharma, Boulder, CO, L. Pieti Opie, Array

BioPharma, Boulder, CO, G. Hingorani, Array BioPharma, Boulder, CO, W. E.

Delaney, Gilead, Foster City, CA, H. Yang, Gilead, Foster City, CA, A. Kaup,

University of Texas Southwestern, Dallas, TX, M. Gale, University of Texas

Southwestern, Dallas, TX, J. Winkler, Array BioPharma, Boulder, CO, J. Josey, Array

BioPharma, Boulder, CO, L. M. Blatt, InterMune, Brisbane, CA

 

Introduction

The standard of care (SoC) for chronic HCV infection, PEG IFN

Α -2 and ribavirin, results in a sustained virologic response in ~50% of patients, demonstrating a clear need for the development of novel therapeutic approaches to treat this disease. We therefore embarked on a

rational drug design campaign to produce inhibitors of the HCV NS3/4 protease. Two lead compounds (ITMN A and ITMN B) emerged from our discovery paradigm with EC50 potencies in biochemical and replicon assays of <2 nM (genotype 1b), liver levels in multiple species predictive of efficacious exposure in humans, and acceptable tolerability. Both of these

compounds have been designated preclinical candidates and are currently undergoing INDenabling toxicological assessment. The activity of both compounds on forms of the NS3/4 protease resistant to two other NS3/4 protease inhibitors, VX-950 and BILN-2016, was examined. Mutation of an alanine at position 156 to a serine (A156S) is a dominant resistance

mutation to VX-950. ITMN A and ITMN B showed IC50s against the A156S NS3/4 protease of 4.5 nM and 2.4 nM, respectively; roughly 1,000-fold more potent than VX-950. Similarly, D168V is a primary resistance mutation to BILN-2061 and ITMN A and ITMN B showed EC50s

against the D168V NS3/4 protease of 10 nM and <2 nM, respectively.

In an effort to understand the molecular basis for the ~50% response rate to the SoC for chronic HCV, a subgenomic HCV replicon that displayed more than a 28-fold reduced sensitivity to the antiviral effects of IFN α-2a was evolved in vitro. This replicon partially evades interferon responses by blocking the activation of interferon regulatory factor-3 (IRF-3) and consequently the downstream expression of a repressor of viral translation (ISG56). ITMN compounds retained their full potency against this interferon “resistant” replicon and furthermore restored expression of ISG56.

 

Conclusion

Pre-clinical candidates ITMN A and ITMN B remain highly active against NS3/4 mutants resistant to VX-950 and BILN-2061. Thus, these compounds may have utility for the treatment of any naturally occurring- or drug induced- HCV quasispecies containing A156S or D168V mutations in NS3/4. In addition, the uncompromised activity of ITMN A and B on an

IFN a-2 insensitive HCV subgenomic replicon implies that these two compounds, and perhaps NS3/4 protease inhibitors in general, may disrupt viral processes that suppress response to interferon treatment.


 

Abstract ID: 65933

Category: JO6: HCV Therapy: Preclinical and Early Clinical Development

The immunomodulator AS101 inhibits HCV replication.

M. Shalita-Chesner, Rabin Medical Center, Petach Tikva, Israel, R. Zemel, Rabin

Medical Center, Petach Tikva, Israel, M. Gal-Tanami, Rabin Medical Center, Petach

Tikva, Israel, B. Sredni, Bar-Ilan University, Ramat-Gan, Israel, L. Bachmatov, Rabin

Medical Center, Petach Tikva, Israel, I. Benhar, Tel-Aviv University, Tel-Aviv, Israel,

R. Tur-Kaspa, Rabin Medical Center, Beilinson campus, Petach-Tikva, Israel

 

Introduction

Current therapeutic options for hepatitis C virus (HCV) infection are limited; therefore, alternative therapeutic approaches are needed. Inhibition of HCV gene expression and replication as well as immunotherapeutic concepts aimed at enhancing the cellular immune response against HCV are being explored. One promising new strategy is the use of immunomodulating agents in combination with IFN and/or ribavirin to treat HCV

patients. The nontoxic immunomodulator ammonium trichloro (dioxoethylene-o,o') tellurate (AS101), first developed by us has been shown to have beneficial effects in diverse preclinical and clinical studies. Most of its activities have been primarily attributed to the direct inhibition of the anti-inflammatory cytokine IL-10. This immunomodulatoryproperty was found to be crucial for the clinical activities of AS101, demonstrating the

protective effects of AS101 in viral-infected mice models. AS101 have also inhibitory effect on different viral catalytic functions such as inhibition of the HIV reverse transcriptase. Phase I clinical trials with advanced cancer patients treated with AS101 showed increased production and secretion of a variety of cytokines, leading to a clear dominance in TH1 responses with a concurrent decrease in the TH2 responses.

 

Aim

The aim of our study was to investigate the inhibitory effect of the AS101 on the catalytic activity of HCV NS3 serine protease in order to evaluate its potential as an HCV therapeutic agent. The effect of AS101 on HCV serine protease activity was analyzed in vitro using the fluorometric assay developed in our laboratory. This effect was further confirmed by our novel high-throughput in-vivo genetic screen. This screen is based on

the concerted co-expression of a reporter gene and recombinant NS3 in E. coli. The effect of AS101 on HCV in-vivo was determined using a cell-based selectable subgenomic HCV RNA Huh-7 replicon system. In this system HCV RNA replication is monitored in intact cells by the enzymatic assay of placental alkaline phosphatase (SEAP) activity secreted.

 

Results

Our study demonstrates that AS101 specifically inhibits NS3 serine protease

catalysis in a dose-dependent manner in the assay system examined. It reaches 70.3 ± 9.5% inhibition at AS101 concentration of 10 mg/ml. Moreover, AS101 specifically inhibited HCV replication as determined in Huh-7 replicon system.

 

Conclusion

Our results suggest that HCV NS3 serine protease is a novel target for AS101 action and of the inhibitory effect of AS101 on HCV replication involve repression of HCV NS3 serine protease.


 

Abstract ID: 67536

Category: JO6: HCV Therapy: Preclinical and Early Clinical Development

Characterization of in vitro selected Con 1 subgenomic replicons resistant to 2’-Cmethyl-cytidine, a potent inhibitor of genotype 1a and 1b HCV polymerases.

S. Rajyaguru, Roche Palo Alto LLC, Palo Alto, CA, S. Le Pogam, Roche Palo Alto

LLC, Palo Alto , CA, V. Leveque, Roche Palo LLC, Palo Alto, CA, H. Kang, Roche

Palo Alto LLC, Palo Alto, CA, H. Ma, Roche Palo Alto LLC, Palo Alto, CA, S. Jiang,

Roche Palo Alto LLC, Palo Alto, CA, K. Klumpp, Roche Palo Alto LLC, Palo Alto, CA,

J. Symons, Roche Palo Alto LLC, Palo Alto, CA, N. Cammack, Roche Palo Alto LLC,

Palo Alto, CA, I. Najera, Roche Palo Alto LLC, Palo Alto, CA

 

 

Introduction

Characterization of in vitro selected Con 1 subgenomic replicons resistant to 2’-Cmethyl- cytidine, a potent inhibitor of genotype 1a and 1b HCV polymerases. The HCV polymerase is an attractive target for the development of new and HCV specific antiviral compounds. 2’-C-methyl-cytidine is a potent inhibitor of both genotype 1b (GT-1b) and GT-1a-polymerase driven replicon replication (IC50 values of 1.0 μ M). The corresponding 5’-triphosphate derivative is a potent and selective inhibitor of native HCV replicase isolated from replicon cells (IC50 value of 0.22

Μ M), and of the recombinant GT-1b and GT-1a HCV polymerase mediated RNA synthesis, (IC50 values of 0.167 μ M for BK NS5B and 0.24 μ M for H77 NS5B). Previous studies have shown that the replicon system allows the selection of replicon variants resistant to HCV inhibitors.

 

Methods

In this study, we used the HCV subgenomic replicon system to select and

characterize HCV variants with reduced susceptibility (>20-fold compared to wild type) to 2’-C-methyl-cytidine.

 

Results

Characterization of resistant replicons by sequence analysis confirmed the presence of amino acid substitution(s) in the NS5B coding region. Site directed mutant transient replicons confirmed the role of the observed mutation(s) in the resistant phenotype. The HCV replicase complex isolated from cells containing mutant replicons as well as the mutant recombinant polymerase-mediated RNA synthesis further confirmed the observed reduced susceptibility to the corresponding 5’-triphosphate derivative. Cross resistance was observed with 2’-C-methyl-adenosine. However, no

cross resistance was observed with the nucleoside analog R1479 or with the non nucleoside, benzothiadiazine- or thiophene carboxylic acid-derivative HCV polymerase inhibitors.

 

Conclusion

This data will allow the optimization of new polymerase inhibitors and their use in combination therapy.


Abstract ID: 67538

Category: JO6: HCV Therapy: Preclinical and Early Clinical Development

An á-glucosidase inhibitor approved for use in type II diabetes has antiviral activity and displays synergy with IFN alfacon-1 in a surrogate system for HCV.

H. Tan, InterMune, Brisbane, CA, S. Seiwert, InterMune, Brisbane, CA, L. M. Blatt,

InterMune, Brisbane, CA

 

Introduction

Bovine viral diarrhea virus (BVDV) belongs to the Flaviviridae family, which includes important human pathogens such as hepatitis C virus (HCV). In addition to being an important bovine pathogen, BVDV is used as a surrogate model for HCV. Both HCV and BVDV contain plus-stranded RNA genomes that encode about nine functionally analogous gene products and replicate through similar mechanisms. The envelope glycoproteins E1 and E2 interact either noncovalently (as in HCV) or through disulphide bonds (as in BVDV) to form a dimer, which has been proposed to be the functional complex present on the surface of mature virions. The envelope glycoproteins of both HCV and BVDV interact with calnexin during productive folding. Both HCV and BVDV have a similar lifecycle, portions of which are not represented in HCV replicon systems. Thus, BVDV represents a suitable model for drug discovery efforts directed toward inhibiting HCV envelope formation and/or function.

 

Miglitol (Glyset®) is an oral α-glucosidase inhibitor used in the management of non–insulin-dependent (type II) diabetes mellitus (NIDDM). Miglitol is a deoxynojirimycin derivative that belongs to an imino-sugar family. Recently, it has been discovered that inhibitors of host endoplasmic reticulum (ER) glucosidase are potent antiviral agents that inhibit replication of a number of viruses.   Glucosidase inhibitors block the first step in glycoprocessing and interfere with glycoprotein folding. We hypothesized that miglitol may have utility in the treatment of HCV by prohibiting formation of the glyco-envelope of the viral particle and ultimately blocking assembly of infectious viral particles.

 

The goals of this study were to test whether miglitol inhibits BVDV replication in vitro and to investigate whether it would provide additional insight into possible synergistic antiviral effects when used in combination with interferon (IFN) alfacon-1, a potent consensus type I IFN approved for use in chronic HCV infection.

 

Materials and Methods

Virus and cells

The cytopathic BVDV strain NADL (ATCC) was used to infect Madin-Darby bovine kidney (MDBK) cells maintained in RPMI 1640 medium (Gibco-BRL) supplemented with 10% horse serum (Sigma, St. Louis, MO) at 37°C in a humidified 5% CO2 atmosphere.

Antiviral assay

MDBK cells were infected in triplicate at a multiplicity of infection (MOI) =

0.01. Virus inoculum was removed, and monolayers were washed twice

with MDBK medium 1 h postinfection. MDBK cells were treated for 48 h with (1) IFN alfacon-1, miglitol, or both, or (2) BILN-2061, miglitol, or both, with a serial dilution. Cells were harvested and RNA was extracted by standard protocols.

 

Viral RNA quantification

Viral RNA was quantified using real-time polymerase chain reaction (PCR;

Prism 7900HT Sequence Detection System, ABI, Inc., Foster City, CA).

BVDV viral RNA was reverse-transcribed using sense primer 5'-GTGACTGCAGGTCGGGTAA- 3', amplified with the same primer and antisense primer 5'-AAATAGTGGCCCTGGCTTCA-3', and detected using a TaqMan® probe the 5' NTR of BVDV(AJ133738). Primer/probe sets were purchased from ABI, Inc. To determine viral RNA concentrations, an absolute standard for viral RNA was obtained by in vitro transcription of a BVDV RNA-encoding DNA and normalized to raw cell count.

Synergy and antagonism analysis

The antiviral effects of drug combinations were evaluated through two different mathematical methods: isobologram analysis and combination index (CI) value using CalcusynTM (Biosoft Inc., Cambridge, UK).

 

Conclusions

·       An approved α-glucosidase inhibitor with a favorable safety profile acts as an antiviral in the BVDV system, a surrogate live virus system for HCV

·       α-Glucosidase inhibitors indirectly target viral structural proteins rather than activities of nonstructural proteins, such  as NS3/4A protease and NS5B polmerase,and are not expected to experience the same mutational pressures as direct antiviral drugs

·       This α-glucosidase inhibitor acts synergistically to augment the pleiotropic activities of IFN alfacon-1 and the direct antiviral activities of an HCV NS3/4A protease inhibitor

·       Triple combination of type I interferon (IFN alfacon-1), α-glucosidase inhibitor, and NS3/4A protease inhibitor showed a very strong synergistic effect

·       Therapeutic regimens with α-glucosidase inhibitors as a component warrant consideration

 


 

Abstract ID: 66361

Category: JO6: HCV Therapy: Preclinical and Early Clinical Development

Immunophenotyping Profile of CPG 10101, a New TLR9 Agonist Antiviral for Hepatitis C.

J. G. McHutchinson, Duke University, Durham, NC, B. R. Bacon, St. Louis University Hospital, St. Louis, MO, S. C. Gordon, Henry Ford Hospital, Detroit, MI, E. Lawitz, Alamo Medical Research, San Antonio, TX, M. Shiffman, Medical College of Virginia, Richmond, VA, N. H. Afdhal, Beth Israel Deaconess, Boston, MA, I. M. Jacobson, Cornell University, New York, NY, A. Muir, Duke University, Durham, NC, S. M. Efler, Coley Pharmaceutical Group, Ottawa, Canada, A. Vicari, Coley Pharmaceutical Group, Ottawa, K. Myette, Coley Pharmaceutical Group, Ottawa, Canada, N. Ahluwalia, Coley Pharmaceutical Group, Ottawa, P. Murzenok, Coley Pharmaceutical Group, Ottawa, G. Lipford, Coley Pharmaceutical Group, Wellesley, MA, T. K. Schmalbach, Coley Pharmaceutical Group, Wellesley, MA, A. M. Krieg, Coley Pharmaceutical Group, Wellesley, MA, H. L. Davis, Coley Pharmaceutical Group, Ottawa, Canada

 

Introduction

CPG 10101 is one of a novel class of drugs, CpG oligodeoxynucleotides, which target Toll-like receptor 9 (TLR9) found on human B cells and plasmacytoid dendritic cells (pDC). It is being developed as an antiviral and Th1 immune enhancer for treatment of chronic infection with the hepatitis C virus (HCV). In two Phase I trials, CPG 10101 was shown to induce/ upregulate certain cytokines and chemokines. Dosedependent effects were demonstrated in both healthy volunteers and HCV infected subjects. Laboratory evaluations in those trials have been extended to immunophenotyping of peripheral blood mononuclear cells (PBMC) to ascertain maturation and stimulatory effects of CPG 10101.

 

Methods

In the Phase Ia trial, forty healthy volunteers were enrolled and assigned to 5 sequentially higher dose cohorts (0.25, 1, 4, 10 or 20 mg); randomized to drug (n=6) or placebo (n=2); given as 2 subcutaneous (SC) single doses, 14 days apart. A 6th cohort received 4 mg twice weekly for 4 weeks. Subjects were evaluated to Day 29 for tolerability, pharmacokinetics, pharmacodynamics, and immunophenotyping. In a subsequent Phase Ib trial, HCV positive patients were randomized to CPG 10101 (n=6 or 7) or control (n=2) over the same range of doses administered twice weekly SC for 4 weeks, or at 0.5 and 0.75 mg/kg once weekly for 4 weeks. Antiviral response (HCV RNA change from baseline) was correlated with the immune effects of CPG 10101.

 

Results

In healthy volunteers, CPG 10101 caused up-regulation of several immunophenotypic markers during the first week after dosing. These included increased CD86 and CD38 on B lymphocytes and increased number of pDC with up-regulation of CD86, all predicted by the mechanism of CpG ODN. Subjects also showed a dose dependent up-regulation of CD69 and CD25 activation markers on NK (CD3-, CD16+CD56+) and NK-T (CD3+, CD16+CD56+) cells. CD14+ cells (monocytes/neutrophils) showed a slight and transient decrease one day post-dosing followed by an increase by 2 days. Most of the cellular markers returned to baseline

levels within two weeks after the first dose. Effects were similar after first and second administrations for a given subject. Analysis of immunophenotyping profiles in HCV subjects is underway.

 

Summary

CPG 10101 activates and matures several types of immune cells in healthy individuals. These effects will be compared with those in HCV infected subjects, and both will be correlated with soluble pharmacodynamic markers of function of these cells, including serum levels of IFN-a, IP-10, 2’5’A-synthetase and TNF-a, which were detected by ELISA assay.


 

Abstract ID: 64147

Category: JO6: HCV Therapy: Preclinical and Early Clinical Development

Treatment of HCV-associated progressive liver disease with Tacrolimus: trial using a mouse model.

K. Koike, Dept of Intern Med, Univ of Tokyo, Tokyo, AL, Japan, K. Moriya ,

Department of Internal Medicine, University of Tokyo, Tokyo, Japan, H. Miyoshi,

Department of Internal Medicine, University of Tokyo, Tokyo, Japan, H. Fujie,

Department of Internal Medicine, University of Tokyo, Tokyo, Y. Shintani, Department

of Internal Medicine, University of Tokyo, Tokyo, Japan, S. Shinzawa, Department of

Internal Medicine, University of Tokyo, Tokyo, Japan

 

Background & Aims

A growing number of evidence has been accumulated showing hepatitis C virus (HCV) infection induces alteration in lipid and glucose metabolisms. While the mechanism underlying such states in the liver is still uncertain, it is suspected that the mitochondria and oxidative stress may play a central role in the pathogenesis of liver diseases in HCV infection. Tacrolimus (FK506), widely used in transplantation, has been supposed to have a capacity to protect the mitochondrial respiratory function, while it has recently shown to be ineffective to HCV replication. The aim of this study was to determine whether or not Tacrolimus protects the function of mitochondria and contributes to improvement of lipid and glucose metabolisms in a mouse model for HCVassociated liver disease.

Methods

Tacrolimus was administered for three months to HCV core gene transgenic

mice, which develop hepatic steatosis, insulin resistance and, finally, liver cancer. As a control, nontransgenic littermate mice were also given Tacrolimus. Levels of plasma glucose and serum insulin and lipid profiles in liver tissues were determined before and after Tacrolimus treatment.

 

Results

In the core gene transgenic mice that were treated with Tacrolimus, steatosis

was improved and lipid profile was changed to a pattern resembling that in normal mice.  Insulin resistance was also improved in the core gene transgenic mice treated with Tacrolimus compared to those without Tacrolimus.

 

Conclusions

·       Tacrolimus reversed the effect of the core protein in lipid and glucose metabolisms in vivo. This result may provide a new therapeutic aid for chronic hepatitis C, in which metabolic abnormalities in lipid and glucose contribute to liver pathogenesis.

·       A long term, low dose Tacrolimus trial is now underway


 

Abstract ID: 66509

Category: JO6: HCV Therapy: Preclinical and Early Clinical Development

Absence of Effect of Pegylated Interferon Alfa-2b on the Pharmacokinetics of Valopicitabine (NM283) in Patients with Chronic Hepatitis C.

X. Zhou, Idenix Pharmaceuticals, Inc., Cambridge, MA, M. Rodriguez-Torres,

Fundacion de Investigacion de Diego, Santurce, E. Lawitz, Alamo Medical Research,

San Antonio, TX, E. Godofsky, Bach & Godofsky, Bradenton, FL, N. Afdhal, Beth

Israel Deaconess Medical Center, Boston, MA, B. Fielman Constance, Idenix

Pharmaceuticals, Cambridge, MA, G. Chao, Idenix Pharmaceuticals, Cambridge, MA, S.

Knox, Idenix Pharmaceuticals, Cambridge, MA, N. A. Brown, Idenix Pharmaceuticals,

Cambridge, MA

 

Background

Valopicitabine (NM283) is a nucleoside analog exhibiting anti-HCV activity via inhibition of viral RNA polymerase, and is currently in phase II clinical

development for the treatment of chronic hepatitis C. In a Phase IIa trial, valopicitabine has shown potent anti-HCV activity in combination with pegylated interferon (PegIFN) a-2a, with greater than 4.5 log10 serum HCV RNA reduction, and no viral resistance detected for study periods up to 6 months. In that context, we are reporting pharmacokinetics (PK) of valopicitabine alone and in combination with PegIFN a-2b.

 

Methods

Eligible treatment-naïve HCV-genotype 1-infected patients (18-65 yrs, with

compensated, chronic liver disease, ALT <5 ×ULN and serum HCV RNA >5 log10 IU/mL) were enrolled in this open-label trial and randomized to receive, at a 2:3 ratio, either valopicitabine monotherapy (n=12) or valopicitabine plus PegIFN a-2b (n=18).  Oral valopicitabine was administered once daily starting with 400 mg from day 1 to day

4, 600 mg from day 5 to day 7 and 800 mg thereafter. PegIFN a-2b was administered by s.c. injection once weekly starting on day 8. Intensive steady-state PK sampling was performed over 8 hours on day 15 in all patients.

 

Results

Values of steady-state PK parameters of valopicitabine, measured as plasma

NM107 (the nucleoside moiety delivered by valopicitabine), were omparable when valopicitabine was administered alone or in combination with PegIFN a-2b (Table 1).  Geometric mean ratio (combination vs. monotherapy) and associated 90% confidence interval were 0.95 (0.83-1.11) for Cmax, 0.90 (0.78-1.05) for AUC0-t, and 0.93 (0.81-1.06) for T1/2, indicating an absence of effect of co-administered PegIFN a-2b on the PK of valopicitabine.

 

Table:  PK parameters of NM107 following dosing with valopicitabine alone and in combination with PegIFN α-2b

 

Treatment

Cmax
(µg/mL)

AUC0-t
(µg/mLxhr)

T1/2
(hr)

Tmax1
(hr)

Valopicitabine

2.9 ± 0.9

15.5 ± 5.0

5.0 ± 2.0

3.0 (2.0-4.0)

Valopicitabine
+ PegIFN α-2b

2.7 ± 0.7

13.8 ± 4.2

4.4 ± 0.8

3.0 (2.0-5.0)

1Median and range

 

 

Conclusions

Co-administration of PegIFN a-2b did not alter the pharmacokinetics of

valopicitabine, providing pharmacokinetic support for combined use of valopicitabine with PegIFN in future clinical trials for the treatment of chronic HCV infection.


 

Abstract ID: 66365

Category: JO6: HCV Therapy: Preclinical and Early Clinical Development

CHARACTERIZATION OF A NOVEL APPROACH FOR HCV IMMUNIZATIONUSING SPLENIC DERIVED DENDRITIC CELLS WITH PHAGOCYTOSED HCVNS5 COATED MAGNETIC BEADS.

S. Gehring, Liver Research Center, Rhode Island Hospital and Brown Medical School,

Providence, RI, S. H. Gregory, Laboratory of Infectious Disease, RI Hospital and Brown

Medical School, Providence, RI, C. Aloman, Liver Research Center, Rhode Island

Hospital and Brown Medical School, Providence, RI, P. Wintermeyer, Liver Research

Center, Rhode Island Hospital and Brown Medical School, Providence, RI, J. R. Wands,

Liver Research Center, Rhode Island Hospital and Brown Medical School, Providence,

RI

 

Background

Dendritic cells (DCs) are able to capture, internalize, and process antigens leading to the potent activation of antigen-specific cellular immunity. The aim of this study was to investigate the capacity of splenic DCs that ingest magnetic beads coated with NS5 antigen to induce HCV cellular immune responses in vivo. METHODS: To expand the DC population in vivo, 10

mg of a human flt3 ligand expressing plasmid (pUMVC3-hFLex) was dissolved in 2 ml saline and injected twice (days 0 and 6) into the tail vein of female Balb/C mice within a 5 second period (hydrodynamic delivery). Splenocytes were incubated for 3 hrs with HCV NS5-coated magnetic beads in serum-free medium.

The cells were harvested and cells that contained beads were purified by

passage over a magnetic column. Non-ingested beads were separated from the purified splenocytes by centrifugation on a 20% Histodenz gradient. The isolated population contained >80% DCs and was used for immunization (2 x 106 cells inoculated in the paw of Balb/c mice). DC expression of the maturation markers CD40, CD80 and CD86 was determined before and after ingestion of NS5- coated beads, showing a significant increase of all maturation markers induced by phagocytosis. The induction of cellular immunity was assessed using a conventional CTL assay, a CFSE-T-cell proliferation assay, intracellular cytokine staining and tumor challenge (with stably NS5 expressing murine myeloma cells).

 

Results

In immunized animals, the CTL activity was increased 3-fold

compared to mock immunized mice. Accordingly, tumor challenge with syngenic SP2/0 myeloma cells transfected with and expressing the NS5 gene showed a significant, (>2-fold reduction) in tumor growth. The number of CD4+IFN-g+ cells was increased >3-fold and the number of CD4+IL-2+ increased >5-fold in the DCNS5- bead immunization group. These results paralleled the proliferative response of splenocytes to NS5 protein obtained from immunized animals with the most significant response in the CD4+ population of DC-NS5-bead immunized animals. Finally, this group displayed a significant humoral immune response to NS5 as well.

 

Conclusion

The use of NS5 coated beads combines three important aspects of dendritic cell based immunization in a single step: targeting of the antigen, enrichment and maturation of dendritic cells. The induction of a strong Th1 biased T cell response makes this approach a promising new tool in therapeutic immunization in chronically HCV infected patients.


Abstract ID: 67582

Category: JO5: HCV: Clinical Trials and Therapeutic Developments

Comparison of African American and Non-African American Patient Sustained VirologicResponse in PEG-IFN Alpha 2 + weight-based ribavirin nonresponders retreated with IFN alfacon-1 + IFN gamma-1b.

C. Leevy, New Jersey Medical School Liver Center, Newark, NJ, C. Chalmers, InterMune, Brisbane, CA, L. M. Blatt, InterMune, Brisbane, CA

 

Introduction

Current treatment of hepatitis C virus (HCV) is effective in achieving a sustained virologic response (SVR) in about 50% of the population, but in African American patients the response rate is substantially lower: 19-26%. 

 

·       Patients who fail current therapy are at risk for disease progression, cirrhosis, hepatic decompensation, hepatocellular carcinoma, and liver transplantation

·       Re-treatment of patients who have failed pegylated interferon alfa-2 plus ribavirin (PEG IFN-α-2 + RBV) therapy results in an SVR of only 4-14%

 

Although all the mechanisms responsible for these reduced response rates have not yet been elucidated, one hypothesis suggests that in African American patients, lack of interferon gamma (IFN-γ) production is associated with nonresponse to IFN-α-2 + RBV.

Effective therapies are needed for difficult-to-treat patients with hepatitis C, such as African Americans, patients with genotype 1 and high viral load, and patients who fail PEG IFN-α-2 + RBV therapy.

 

Rationale

Combining Type I and II Interferons Holds Promise for the Treatment of Chronic HCV

·       Consensus IFN (IFN alfacon-1) is a bioengineered, nonnaturally occurring type I IFN that displays 10- to 100-fold higher biological potency when compared with naturally occurring type I IFNs

·       We have demonstrated that antiviral and immunologic effects are enhanced when IFN alfacon-1 and IFN-γ 1b are combined in invitro systems

·       Clearance of acute HCV infection requires a potent IFN-γ response

·       Clearance of HCV in chronically infected patients treated with IFN-α requires a shift from a dominant Th2 to Th1 response (mediated by IFN-γ)

·       RBV induces the production of Th1 cytokines (e.g., IFN-γ)

·       IFN-γ reverses HCV core negative effects on STAT phosphorylation

 

Objective

This retrospective case series examined the safety and efficacy of re-treating patients who did not respond to PEG IFN-α-2 + RBV therapy with a combination of a bioengineered type I interferon (IFN alfacon-1) and a recombinant type II interferon (IFN-γ 1b). Specifically, the objective of this analysis was to compare the efficacy and safety of treatment (tx) of African American patients with non–African American patients.IFN-γ has its own intrinsic antiviral activity

 

Study Design

This study is a retrospective review of patients treated with PEG IFN-α-2 + RBV who had < 2 log10 reduction in HCV RNA (nonresponders).  Therapy was switched to a combination of IFN alfacon-1 15 µg administered subcutaneously (SC) daily (QD) and IFN-γ 1b 50 µg SC 3x/wk (TIW) for 48 wk. SVR was defined as having undetectable HCV RNA 24 weeks after the last dose.

A total of 50 patients were treated: 20 African Americans and 30

non–African Americans.

 

Efficacy

Comparison of Baseline Characteristics by Racial Group (N = 50)

 

 

 

Safety

Comparison of Baseline Characteristics by Racial Group (N = 50)

Efficacy

·       Patients had rapid reduction in ALT after initiation of therapy

·       14 patients (28%) had reductions in WBC (ANC < 0.75 x 109) that required use of G-CSF

·       Dosing was temporarily interrupted for one patient; there were no dosing reductions for other patients

·       Neutropenia was manageable with G-CSF and did not require dose reductions

 

 

Summary

·       Only one patient had dosing temporarily interrupted; no one had a dose reduction

·       Reductions in ANC were well controlled with filgrastim

·       Hemoglobin levels returned to normal during treatment without the use of erythropoietin

·       34% of the patients (overall) who were nonresponders to PEG IFN-α-2 + RBV achieved an SVR after therapy with IFN alfacon-1 + IFN-γ 1b

·       African American and non–African American nonresponder patients had a similar rate of on-treatment virologic response and SVR after therapy with IFN alfacon-1 + IFN-γ 1b

 

Conclusion

·       The combination of IFN alfacon-1 (15 µg QD) + IFN-γ 1b (50 µg TIW) was generally well tolerated in this retrospective review of patients with chronic HCV

·       Reductions in ANC were well controlled with filgrastim and patients’ hemoglobin levels returned to normal during treatment

·       African Americans and non–African Americans had similar rates of SVR

·        Adding IFN-γ 1b to IFN alfacon-1 in the re-treatment of African  American patients who have failed to respond to PEG IFN-α-2 + RBV may emerge as an important treatment option

·       Further study is warranted in a larger patient population

 


Antiviral Response of HCV Genotype-1 to Consensus Interferon and Ribavirin Compared with Pegylated Interferon and Ribavirin 

M. Sjogren, Walter Reed Army Medical Center, Washington , DC, R. Sjogren, Kaiser Permanente Mid Atlantic States, Falls Church, VA, M. F. Lyons, Tacoma Digestive Diseases Center, Tacoma, WA, M. Ryan, Digestive & Liver Disease Specialists, Norfolk, VA, J. Santoro, Atlantic Gastroenterology Associates, Egg Harbor Township, NJ, C. Smith, Minnesota Gastroenterology, St. Paul, MN, K. R. Reddy, University of Pennsylvania GI Research, Philadelphia, PA, H. Bonkovsky, University of Connecticut Health Center, Farmington, CT, B. M. Huntley, Walter Reed Army Medical Center, Washington , DC, L. Ibarra, InterMune, Inc., Brisbane, CA, S. Faris-Young, Intermune, Inc., Brisbane, CA

 

 

Background:

Over 50% of patients treated with genotype-1 chronic HCV infection fail to achieve a sustained virologic response (SVR). Recent studies have shown that consensus interferon (CIFN) in combination with ribavirin (RBV) may be particularly active against genotype-1 HCV infection.

 

Aim:

Proof of concept study to compare treatment with CIFN/RBV to pegylated interferon (PEGIFN) and RBV in achieving SVR. This was a prospective randomized clinical trial where treatment-naïve HCV genotype-1 subjects received either CIFN 15 mcg TIW and weight-based generic ribavirin (Ribasphere) (group 1) or PEG-Intron 1.5 mcg/kg/week and weight based ribavirin (Rebetol), (group 2).  The secondary aim of the study was to assess differences in adverse effect profiles between both treatment groups with emphasis on mental depression using the following scoring systems:

·       Back Depression Inventory II (BDI-II)

·       Center for Epidemiological Studies Depression Scale (CESD)

·       Fatigue Severity Scale (FSS)

 

Methods:

59 subjects were enrolled, 30 in group 1 and 29 in group 2. Treatment lasted 48 weeks if HCV RNA was undetectable at week 24, otherwise drugs were discontinued. SVR was determined at week 72. Safety laboratory tests and adverse events (AE) assessments were done monthly. To date all subjects have completed week 48 and 56 subjects (6 subjects, of which 2 received PEG IFN-a-2b/RBV and 4 received CIFN/RBV, discontinued early and were excluded from the analysis) have finished week 72.  

 

Patient Characteristics

 

Results

The SVR are listed below:

Average Depression Scores on BDI and CESD in Both Treatment Groups

·       Depression measures BDI and CESD had moderate-to-strong positive correlation (p=0.5)

·       Trends of depression and fatigue for CIFN/RBV and PEG IFN- 2b/RBV regimes are distinct, with adverse effects peaking at Weeks 4 and 36, and Week 24, respectively.

 

Average Fatigue Scores on Fatigue Severity Scale in Both Treatment Groups

·       Higher fatigue scores parallel higher depression scores.

·       =p=Non-significant; scores on fatigue scales were not significantly different in both treatment regimes at any time during the treatment.

 

Summary

·       CIF 15 ug TIW + weight-based ribavirin elicits a comparable response to PEG IFN-a-2b + weight-based ribavirin in HCV genotype-1 patients.

·       All subjected treated with CIFN/RBV that had an undetectable HCV RNA at Week 48 because sustained responders

·       The safety profile for both combination regimes was similar

 

Conclusion:

·       CIFN/RBV combination therapy elicited a comparable SVR to PEGIFN/RBV in previously untreated subjects with genotype-1 chronic HCV.

·       There were no relapsers among the CIFN/RBV treated group. The AE profile for CIFN/RBV demonstrated less neutropenia and dose reductions.

·       It is possible that daily CIFN + weight-based RBV may achieve higher SVR in subjects with HCV genotype 1 infections as suggested by studies by Kaiser and Rustgi

·       A larger clinical trial for genotype-1 chronic HCV infection utilizing CIFN and ribavirin is warranted.